This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Homologous recombination (HR) plays a key role in repairing double-strand breaks in DNA. Recent studies indicate that defects in HR and double-strand break repair cause several human cancer-related syndromes, suggesting the importance of HR and related DNA repair pathways in genome integrity. In this proposal, we will focus on determining regulation of a "network" composed of HR and downstream enzymes, by applying a combination of biochemical, molecular, and genetic approaches in yeast. Our preliminary data suggest that there are multiple HR pathways, which are distinguished by Sgs1, Mus81 and RNase H2. Our challenge is determining the differences between multiple HR pathways. The other main project is determining role of RNase H2 in "recombination-mediated DNA repair". RNase H2 is a minor component for Okazaki fragment processing in DNA replication. Mutations of RNase H2 in human cause Aicardi-Goutieres syndromes, which is a fatal neurological disease. Our previous study indicated that RNase H2 plays a significant role in DNA repair in parallel with Sgs1 and Mus81, which both function downstream of HR. We will define the mechanism how RNase H2 functions downstream of HR. Based on our hypothesis that RNase H2 might make a larger complex for DNA repair, native RNase H2 supercomplex will be purified, assayed for nuclease activity and substrate specificity. The other project is defining in vivo function of Mus81. Our previous study provided novel functions of Mus81 in vivo, such as roles in checkpoint control and in rDNA expansion/contraction. We will identify other functions of Mus81 in vivo, by seeking protein modification such as SUMOylation and ubiquitylation. The results of these experiments will provide new insights into the maintenance of genome stability controlled by a network of HR and related enzymes and they will be applied for understanding of maintenance of genome stability in human cells.